Q: Trouble with Kindergarden Ligation (SspBI/ApaI)

David B. Knaebel, knaebel at draco.clarkson.edu
Mon Sep 22 11:44:12 EST 1997

Wolfgang Schechinger wrote:
> Hi you all molbio gods and experts!
> to introduce the mutation, the PCR fragment is trated in the same way
> to get sticky ends. The fragments the are gel purified and ligated
> (T4 ligase, overnight at room temp). Then: either no colonies or as
> much as on the control plate (excised vector only).
> What's happening here? Actually this shouldn't be a problem for
> molbio beginners in their first lesson. Have you any ideas?
> Could it be that four bases are not enough for the enzymes to find
> and cut their sites?
> All input is welcome!

I had a similar problem with two sets of primers that had XhoI and XbaI
sites at the ends.  In a research project, the first set did not work,
but I got some clones because of some odd rearrangements that were
detected by sequence analysis. To remedy this, I increased the numbers
of GC's  at the 5' ends, but had very little improvement. I am going to
try this set of primers once more for directional cloning, but am also
going to use a pCR script type system as a backup. (The students always
like it when their cloning works!)

Dave Knaebel

Biology Department
Clarkson University
Potsdam NY 13699-5805
(315) 268-2391
(315) 268-6610 [fax]
knaebel at draco.clarkson.edu

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